1. Field of the Invention
The present invention relates to methods for producing a polypeptide in a bacterial cell.
2. Description of the Related Art
Bacilli are well established as host cell systems for the production of native and recombinant proteins. However, Bacillus host cells with the desirable traits of increased protein expression may not necessarily have the most desirable characteristics for commercial use.
Conventionally, maximal expression of a gene contained in a Bacillus cell is achieved by amplifying in the chromosome an expression cassette containing a single promoter operably linked to a gene of interest and an amplifiable selectable marker gene, e.g., an antibiotic resistance marker. The amplification leads to the production of multiple copies of the expression cassette and the selectable marker gene in the chromosome.
However, there are disadvantages associated with this approach. For example, it may not be possible to achieve saturating levels of mRNA by amplifying genes driven by single promoters. Also, the production of multiple copies of the expression cassette and the selectable marker gene in the chromosome of a cell may not be stable. Furthermore, the removal of the selectable marker genes without also losing the expression cassettes may not be technically feasible.
Ichikawa et al. (1993, FEMS Microbiological Letters 111: 219–224) disclose the extracellular production of cholera toxin B in Bacillus brevis containing an expression-secretion vector with multiple promoters which mediate the expression of the gene encoding the mature cholera toxin B.
Agaisse and Lereclus (1994, Molecular Microbiology 13: 97–107) disclose a structural and functional analysis of the promoter region involved in the full expression of the cryIIIA toxin gene of Bacillus thuringiensis. WO 94/25612 discloses an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of the cryIIIA gene which increases expression of the gene.
Hue et al. (1995, Journal of Bacteriology 177: 3465–3471) disclose a 5′ mRNA stabilizer sequence which stabilized several heterologous RNA sequences when present at the 5′ end and increased expression of downstream coding sequences several-fold in Bacillus subtilis. 
It is an object of the present invention to provide improved methods for producing a polypeptide in a Bacillus strain.